45.Isolation, Purification and kinetics of deoxy Ribonucleic acid at differen...
02.Production of Amylase in Aspergillus oryzae
1. EIOJOURNAL,VOL.lO NO.1 & 2,179.185 JUNE & DECEMBER,l99S
PHODUCTION OF AMYLASE IN ASPE'RGILLUS ORYZAE
B. Annadurai, V. Prabahkaran, S. Moharnrned Faruck
and F. lrulkumaran
Centre oI Biotechnology, Department of Biochemistry, C. Abdul Hakeem College,
Melvisharam- 632 509, Vellore District.
ABSTRACT
Out of 30 rnicroorganisms isolated and was grown in yeastarch medium.The medium
in which the microbe grows determines the type and quantities sf the extra cellular
enzyrnes.
Out of various fungi and bacteria grown in the medium Aspergillus spp. and Yeast
spp. are growing very well in this medium on the 4th day of the culture. Amidst various
organisms Aspergillus sryzae produces the extra cellular amylase more when compare
to all other species.
INTRODUCTION
Arnylase degrades the starch and is of two types (Myrback and Neumulkar, 1950).
AIpha amylases (endoamylase E.C.3.2.1.1.) are distributed in animals, microorganisms
and plants which attack the alpha 1,4 glucosidic bonds of amylose and yields dextrin
and maltose residues. Beta-amylase (E.C.3.2.1.3) are known as hexo amylases (Abdullah,
et. al 1965) are saccharifing amylases release successive maltose units from the non
reducing end of the polysaccharide chain by hydrolyis of alpha - 1,4 glucon linkages
(Myrback and Neumuller, 1950). Amylases are widely produced by fungi and
microorganisrns {Redfern, 1950, Caldwell and Adams 1951 , Schwirnmer 1951 , and Adams
1953). illearly 130 species are able to assimilate starch (Earnett etal 1984). Bacterial and
Mold arnylase clue to their industrial importances extensively much studied (Fogarty W.
M. and Kelly C.1 1983). lt is potentially usefull for single step ethanol production and
conversion of starehy waste material into single cetl protein (Siso et. al., 1988 and *ills
et. al. 1987).
The pap*r r{eals with the produetion of arnylases in differeni microorganisms in the
medium for Sarge scale production.
IVTTTEH IALS & FJ D frI! ETH CI DS
STRAIN:-
Varous strains of fungi, bacteria were iselated frorn the petridishes eontaining PDA
medium.The aLltoclaveri media after cooling were kept at different piaces io get various
strains of tungi and bacteria.Type cultures were obtained fronr Centre of advance study
in Botany, Universlty of Madras and Department of Eiotechn*logy, eentral leather research
institute, Ghennai and other strains cbtained frorn thB slJbcultures of loeal Departments
(179)
2. ANNADURAIet. al.
and Colleges.
The pure cultures were grown on a solid medium of the foltowing cornposition (g/l).
Yeast extract 3.0
Malt extract 3.0
Peptone 5.0
Starch
Agar
pH
LlOUlD MEDIUM:-
The amyfase production medium is based of Bajpai and Sharma (1989) with the
following compoSition (g/l)
Starch
Yeast extract 3.0
10.0
30.0 -
4.5 to 5.0
4.0
0.85
0.15
0.50
0.10
0.10
4.5 to 5.0
10.0
Peptone
KH2PO4
K2ltPO4
MgSo4.7H2)
NaCI
CaCl2.2H2Q
pH
The inoculum was prepared by suspending a loop full of 48 hour old stock culture
into 10 ml of sterlized distilled water to acheive an O.D" ranging f rom 0.5 to 1.0 at 620 nrn.
MYCELIA!- DRY WEIGHT DETERMINATION :-
50 ml of the culture medium was dispensed in each 150 ml Erlenmeyer flask and
sterilized by autoclaving. Starch was autoclaved separately and aseptically added in the
medium after sterlization.The f lask were inoculated with 1 rnl of cell suspension (prepared
from 5 day old culture) containing approximately 106 cells and incubated at 32 + 1 c
without shaking.lt is allowed to grow for 5 days.The contents were filtered through pre
weighed whatmann filter paper No.-1 and the culture filtrate thus obtained was used as
crude enzyn'le source for a and b amylases.
ESTIMATION OF ALPHA AMYLASES :-
Alpha amylases was measured colorimetrically by the rnethoc8 of Sandhu ef. a/"
(1S0)
3. BIOJOTJRNAL, JUNE & DECEMBER' 1998
(1gg7) using 3 ml1% Soluble starch solution prepared in 0.05 M citrate phosphate buffer
ipff s.zl incl-rbated with 1 mt of enzyme sample at 37 C. for 15 minutes' Amylase activity
is defined as the log decrease in 0.D. at 550 nm in 15 minutes of incubation'
ESTIIJIATION CF BETA, AMYLA$E :.
Beta amylase activity is measured by the method of Bernfeld (1955)'The reaction
mixture consist of 1 ml of crude nrl f iltrate and 3 rnl of 1% starch (in 0.05 M citrate phosphate
buffer pH 5.2) was incubatecl for 3 minutes at 37 C. fhe reaction was stopped by the
aclclition of 3 ml of 1% ciinito saiicylic acid (DNSA). Reagent tubes were then placed in
boling water for 5 minute and the absorbance was measured at 540 nm against a blank
prepa?ed with hoiled culture filtrate. Maltose was used as the standard. Beta arnylase
unii is expressed as rnicro mcles of maltose produced by milligram of protein per minute'
EST|MATION GF FHOTfiIN :-
Protein was measured by the rnethod of Lowry et. al. (1951) using Bovine serum
albumin as stanelard.
FIESULTS AND DISCUSSIONS
l-able -i reveals the ge.ol,rth of micro-organisms inY-N based medium the production
oi alp{"ra and beta anTylase activity was estirnated. out of various species Alternaria spp,
Aspergilius species, Bacillus species, Rhizopus spp., Yeast species shows maximal
grorvtil and ainylase produclion. Out of all the species Aspergillus shows optimum
amylase activitY.
Table - 1
GHSWTij CF MIICfiCORGANISIIII AI,ID PRODL,CTION OF AM'/LASE :-
a-amylase
activity
B-amylae
Achrornobacten sPP.
Achromabaater
Alternari aibugo
Alternari solani
Alternaritenuis
Alternarl triticina
Alternari triticola
Ascochyta Pisi
Aspergillus aureus
Aspergillus citreus
Aspergil!us flavus
Aspergillus f uniculosis
+
NS
+
+
+
+
+
NS
++
++
++
++
NS
NS
NS
NS
NS
NS
t{s
NS
+
+
+
+
(181)
5. 50.
51.
52.
cJ.
54.
l;G
56.
57.
5t,
EA
AA
t)t.
IJZ-
oJ.
64.
65.
tr6-
or"
Fencillium spp"
Pencillium spp" I
Pencillium spp" 
